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Previous Article | Next Article 
Journal of Clinical Microbiology, October 1999, p. 3402-3404, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Elimination of Bacterial DNA from Taq
DNA Polymerases by Restriction Endonuclease Digestion
Nora M.
Carroll,
Peter
Adamson, and
Narciss
Okhravi*
Department of Clinical Ophthalmology, The
Institute of Ophthalmology, London EC1V 9EL, United Kingdom
Received 2 April 1999/Returned for modification 4 June
1999/Accepted 6 July 1999
 |
ABSTRACT |
The incidence of false positives due to the presence of bacterial
DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a
restriction enzyme to destroy the ability of contaminating sequences to
act as templates for a nested PCR which uses primers based on the 16S
rRNA genes. The method was used prior to a PCR that amplified 10 fg of
bacterial DNA. This method can be readily adapted to suit other
sensitive PCRs required for clinical applications.
| |
TEXT |
Due to its ability to exponentially
amplify regions of DNA, the PCR has the potential to be used as a
diagnostic tool. It has been used in the detection and identification
of a wide range of bacterial species using oligonucleotide primers
based on the conserved regions of rRNA sequences. The detection of
organisms which are difficult to cultivate has been improved by the use of this technique (1, 5, 8), as it relies on the presence of DNA and not the viability of the organism. The disadvantage of
primers with broad specificity is the concomitant amplification of
contaminating DNA, which gives rise to false-positive results. The elimination of false positives is an essential prerequisite to the
development of PCR protocols for use in a clinical setting. This
objective has been partly achieved by rendering PCR amplicons unsuitable for reamplification and also by the elimination of contaminating template DNA in PCR reagents. The presence of bacterial DNA in preparations of Taq DNA polymerase is well
established (3, 4). Several methods have been used to
eradicate this contamination (6, 7, 9), but none were found
to be 100% effective. Diagnosis by culture is especially difficult
with clinical samples when a limited volume is available. A set of
nested primers was developed for the detection of bacteria which were
capable of detecting 10 fg of bacterial DNA (unpublished observation). However, these primers were found to be of limited use, as every reagent control which was subjected to two rounds of amplification gave
rise to a product of the size expected for a bacterial template, although none was added. In this paper, a method is outlined for the
removal of contaminating DNA sequences from Taq DNA
polymerase preparations which can be easily applied to other PCR assays.
Pretreatment of Taq DNA polymerase to remove
contaminating bacterial DNA.
Prior to PCR amplification, the
water, buffer, MgCl2 and Taq DNA polymerase
components were mixed and incubated for 30 min at 37°C with 1.0 U of
Sau3AI (Boehringer Mannheim) per U of Taq DNA
polymerase. The restriction enzyme was inactivated by incubation at
95°C for 2 min, following which the deoxynucleoside triphosphates, primers, and template DNA were added and PCR amplification commenced. Taq DNA polymerase for the second round of amplification was
used as supplied. Details of the two primer pairs used in this study appear in Table 1. PCR mixtures contained
60 µM each deoxynucleoside triphosphate (Pharmacia), 0.3 µM each
primer, 3.0 mM Mg2+, and 1 U of various Taq DNA
polymerases in 25 µl. First-round amplifications were conducted with
2.5 pmol each of primers 16SF and 16SR, with an initial denaturation at
95°C (3 min) and cycling as follows: 95°C for 10 s, 54.2°C
for 10 s, and 72°C for 15 s for 30 cycles (Genius Thermal
Cycler; TECHNE). The second-round amplification was carried out as was
the first, except that the Mg2+ concentration was 2.5 mM
and 5 pmol of each primer was used. One microliter of the product from
the first-round amplification was amplified with primers NF and NR as
follows: denaturation at 95°C (3 min) and cycling at 95°C for
7 s, 60°C for 7 s, and 72°C for 10 s (30 cycles).
Reagent controls from the first round were always subjected to a second
round of amplification to control for contamination.
Amplicons were resolved on a 1% agarose-Tris-acetate-EDTA gel,
visualized by using ethidium bromide under UV illumination, and
recorded by using the UVP gel documentation system (UVP Ltd.). Samples
for Southern analysis were purified by using Qiaquik PCR purification
kits (Qiagen) and slot blotted onto Hybond N+ (Amersham).
Probes for Southern hybridizations were 3' labelled and detected by
using the ECL 3' labelling kit (Amersham).
Contaminating sequences in Taq DNA polymerase are readily
amplified by using universal bacterial primers based on ribosomal genes. All assays to check for DNA in Taq DNA polymerase
were carried out by using fresh reagents to rule out the possibility of
exogenous contamination. The level of contamination was insufficient to
give a detectable product after one round of PCR but was easily detected in all preparations after two rounds of amplification (Fig. 1). Amplitaq LD routinely
demonstrated the least PCR product production following nested
amplification. Prior to first-round PCR amplification, Amplitaq LD was
treated with Sau3AI as already described. It can be seen
from Fig. 2 that use of very high levels of Sau3A1 (4 U/U of Taq DNA polymerase) as a
pretreatment resulted in reduced product formation from 20 ng of
template DNA. Lower levels of Sau3AI achieved the aim of
avoiding false positives while maintaining an acceptable level of
sensitivity after two rounds of PCR amplification. Treatment of the
Amplitaq LD prior to the second round of amplification was not
performed, as the subsequent sensitivity of detection was compromised
(data not shown). Southern hybridization of the negative controls
in Fig. 2B showed that the untreated negative control was the only
sample that gave a positive signal (data not shown). As can be
seen in Fig. 3, inclusion of 1 U of
Sau3AI/U of Amplitaq LD allowed the amplification of 10 fg
of bacterial DNA, illustrating that this method does not compromise the
sensitivity or specificity of this PCR.
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FIG. 1.
Nested PCRs using primer pairs 16F plus 16R and NR plus
NF amplify a product in the absence of an added template. Lanes 1 to 5 were amplified by using Amplitaq LD (Perkin-Elmer, Cheshire, United
Kingdom), lanes 6 to 10 were amplified with Amplitaq (Perkin-Elmer),
and lanes 11 to 15 were amplified with Taq DNA polymerase
(Stratagene, Amsterdam, The Netherlands). Lanes 1, 6, and 11 were
positive controls for the outer PCRs; lanes 2, 7, and 12 were reagent
controls for the outer reaction; lanes 3, 8, and 13 were positive
controls for the nested reaction; lanes 4, 9, and 14 contained 1 µl
of the first-round reagent control amplified with the nested primers;
and lanes 5, 10, and 15 were reagent controls for the nested PCR. Lane
16 contained the molecular size marker (Promega, Wis.).
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FIG. 2.
Effect on product amplification of treating
Taq DNA polymerase with decreasing amounts of
Sau3A1. Positive reactions contained 20 ng of
Escherichia coli NTCC 11151 DNA. Negative reaction mixtures
contained no added template. (A) First round of amplification with
primers 16F and 16R. Lanes 1, 3, 5, 7, 9, 11, and 13 were positive
reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of
Sau3AI, respectively. Lanes 2, 4, 6, 8, 10, 12, and 14 were
negative reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lane 15 contained the molecular
size marker (GIBCO, Paisley, Scotland). (B) Second round of
amplification with primers NF and NR. Lanes 1 to 7 contained 1 µl of
the negative reaction mixtures from round 1 that had been treated with
4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively.
Lane 8 contained the positive control for the amplification, and lane 9 contained the reagent control. Lane 10 contained the molecular size
marker (GIBCO).
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FIG. 3.
Sensitivity of nested PCR amplification of E. coli NTCC 11151 DNA using primer pairs 16F plus 16R and NF plus
NR. The template was E. coli NTCC 11151 DNA purified as
described in the text. Lanes 1 to 8 contained the products of the
amplification of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg of
DNA or no DNA using primers 16F and 16SR and Amplitaq LD that had been
pretreated with Sau3AI. Lane 9 contained the 1-kb size
marker (Promega). Lanes 10 to 17 contain the products of the
amplification of 1 µl of each of the first-round samples with primers
NF and NR. Lane 18 contains a reagent control for the second round of
amplification.
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|
Taq DNA polymerase is contaminated with bacterial DNA
(3, 7). Amplitaq LD from Perkin-Elmer, an ultrapure
preparation, is guaranteed to contain less than 10 copies of bacterial
16S rRNA gene sequences per 2.5-µl aliquot, which was sufficient to produce a false-positive result under our conditions of amplification. To eliminate this source of contamination, a pretreatment protocol was
developed which successfully rendered this DNA unamplifiable while
maintaining the sensitivity of the reaction. Additional criteria for
choosing Sau3A1 were its ease of inactivation by heat and
its wide availability. Several investigators have reported methods for
reducing or eliminating the amplification of contaminating DNA
sequences in Taq DNA polymerase. These included pretreatment of Taq DNA polymerase with DNase I or purification on a
CsCl2 density gradient (1-6). DeFilippes
(2) reported that false positivity was reduced by the use of
restriction endonucleases but did not address the question of
contamination of the Taq DNA polymerase itself. The large
amounts of enzyme used and the long incubation time made his procedure
unwieldy, expensive, and impractical for routine use.
The method described in this paper can be easily incorporated into
current PCR protocols, as it utilizes an enzyme that is active in the
PCR buffer, reducing the amount of sample handling and opportunities
for sample contamination. In addition, the short reaction time merely
extends the total time required for the PCR by 30 min. In a clinical
context, this protocol will allow confident reporting of positive
results and its extreme sensitivity will also allow the use of negative
PCR results. This is significant, as clinical diagnoses are often
complicated by immune-mediated processes which can appear
indistinguishable from infection. However, as this technique is used to
remove contaminating sequences only from Taq DNA polymerase,
it is no substitute for general cleanliness and the usual
precautions in the PCR laboratory, i.e., separation of pre- and
post-PCR areas.
| |
ACKNOWLEDGMENTS |
N.C. was supported by Oclyx Ltd. P.A. was supported by Fight for
Sight. N.O. was supported by Wellcome Vision Research Fellowship 045203 and locally organized research funds from Moorfields Eye Hospital (221 and 271).
We thank Susan Lightman, Department of Clinical Ophthalmology, The
Institute of Ophthalmology, for the opportunity to undertake this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Ophthalmology, The Institute of Ophthalmology, Bath St.,
London EC1V 9EL, United Kingdom. Phone: 0171-608-6872. Fax:
0171-608-6931. E-mail:
nokhravi{at}menu.hgmp.mrc.ac.uk.
Present address: Department of Medical Biochemistry, University of
Stellenbosch, Tygerberg 7505, South Africa.
| |
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Journal of Clinical Microbiology, October 1999, p. 3402-3404, Vol. 37, No. 10
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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